Method for drug delivery to the pulmonary system

ABSTRACT

Drug delivery to the pulmonary system has been achieved by encapsulation of the drug to be delivered in microparticles having a size range between 0.5 and ten microns, preferably in the range of two to five microns, formed of a material releasing drug at a pH of greater than 6.4. In a preferred embodiment, the drug delivery system is based on the formation of diketopiperazine microparticles which are stable at a pH of 6.4 or less and unstable at pH of greater than 6.4, or which are stable at both acidic and basic pH, but which are unstable at pH between about 6.4 and 8. Other types of materials can also be used, including biodegradable natural and synthetic polymers, such as proteins, polymers of mixed amino acids (proteinoids), alginate, and poly(hydroxy acids). In another embodiment, the microparticles have been modified to effect targeting to specific cell types and to effect release only after reaching the targeted cells.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. Ser. No. 10/211,215 filed onAug. 2, 2002, by Solomon S. Steiner, Robert Feldstein, Huiling LiamChristopher A. Rhodes, and Gregory S. Shen, which is a continuation ofU.S. Ser. No. 08/441,378 filed on May 15, 1995.

BACKGROUND OF THE INVENTION

This invention is generally in the area of drug delivery systems and isparticularly related to methods of delivery to the lungs and othercomponents of the pulmonary system.

Many drugs are delivered to the lungs where they are designed to have aneffect on the tissue of the lungs, for example, vasodilators orsurfactants, or within the bronchi, for example, a bronchodilator, or ona tissue within the lung, for example, a chemotherapeutic agent. Otherdrugs, especially nucleotide drugs, have been delivered to the lungsbecause it represents a tissue particularly appropriate for treatment,for example, for genetic therapy in cystic fibrosis, where retroviralvectors expressing a defective adenosine deaminase are administered tothe lung. Other drugs which have been used with limited success due todifficulties in administration include vaccines, especially for flu andother respiratory illnesses, where the immune cells of the lung are thetarget.

Advantages of the lungs for delivery of agents having systemic effectsinclude the large amount of surface area and ease of uptake by themucosal surface.

It is very difficult to deliver drugs into the lungs. Even systemicdelivery has limitations, since this requires administration of highdosages in order to achieve an effective concentration within the lungs.

Most drugs now are administered using a dry powder or aerosol inhaler.These devices are limited in efficacy, however, due to problems intrying to get the drugs past all of the natural barriers, such as thecilia lining the trachea, and in trying to administer a uniform volumeand weight of powder.

It is therefore an object of the present invention to provide animproved composition for administration of drugs to the pulmonarysystem.

It is a further object of the present invention to provide a compositionfor controlled pulsed or sustained administration of the drugs to thepulmonary system.

SUMMARY OF THE INVENTION

Drug delivery to the pulmonary system has been achieved by encapsulationof the drug to be delivered in microparticles having a size rangebetween 0.5 and ten microns, preferably in the range of one to fivemicrons, formed of a material releasing drug at a pH of greater than6.0, preferably between 6.4 and 8.0. In a preferred embodiment, the drugdelivery system is based on the formation of diketopiperazinemicroparticles which are stable at a pH of 6.4 or less and unstable atpH of greater than 6.4, or which are stable at both acidic and basic pH,but which are unstable at pH between about 6.4 and 8. Other types ofmaterials can also be used, including biodegradable natural andsynthetic polymers, such as proteins, polymers of mixed amino acids(proteinoids), alginate, and poly(hydroxy acids). In another embodiment,the microparticles have been modified to effect targeting to specificcell types and to effect release only after reaching the targeted cells.

In the most preferred method of manufacture, the microparticles areformed in the presence of the drug to be delivered, for example,proteins or peptides such as insulin or calcitonin, polysaccharides suchas heparin, nucleic acid molecules, and synthetic organic pharmaceuticalcompounds such as felbamate. The diketopiperazine microparticles arepreferably formed in the presence of the drug to be encapsulated by: (i)acidification of weakly alkaline solutions of a diketopiperazinederivative that contains one or more acidic groups, (ii) basification ofacidic solutions of a diketopiperazine derivative that contains one ormore basic groups, or (iii) neutralization of an acidic or basicsolution of a zwitterionic diketopiperazine derivative that containsboth acidic and basic groups. The size of the resulting microparticlescan be controlled by modifying the side-chains on the diketopiperazine,the concentration of various reactants, the conditions used forformation, and the process used in formation. The diketopiperazines canbe symmetrically functionalized, wherein the two side-chains areidentical, or they can be unsymmetrically functionalized. Both thesymmetrically and unsymmetrically functionalized diketopiperazines canhave side-chains that contain acidic groups, basic groups, orcombinations thereof. Methods that can be used with materials other thanthe diketopiperazines include spray drying, phase separation, solventevaporation, and milling, although the latter does not typically yielduniform diameters and can lead to erratic release profiles, which arenot desirable. In the preferred embodiment, the microparticles areadministered by the use of a dry powder—breath activated—compressed airinhaler.

Examples demonstrate the administration of calcitonin encapsulated intwo micron diameter diketopiperazine microparticles to the pulmonarysystem of sheep.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a prospective view of a dry powder—breath activated—compressedair, manually operated inhaler.

FIGS. 2 a and 2 b are graphs of the pharmacokinetics of salmoncalcitonin (sCT) (100 micrograms/kg) administered to sheep byinstillation into the lung (open circles) compared with administrationby subcutaneous injection (closed circles), measured as plasma sCTconcentration (pg/mL) over time (hours), with FIG. 2 b representing thesame data as a semi-logarithmic plot.

DETAILED DESCRIPTION OF THE INVENTION

I. Systems for Pulmonary Administration.

Microparticles having a diameter of between 0.5 and ten microns canpenetrate the lungs, passing through most of the natural barriers. Adiameter of less than ten microns is required to bypass the throat; adiameter of 0.5 microns or greater is required to avoid being exhaled.

A. Polymers for Forming Microparticles.

A number of polymers can be used to form the microparticles. As usedherein, the term “microparticles” includes microspheres (uniformspheres), microcapsules (having a core and an outer layer of polymer),and particles of irregular shape.

Polymers are preferably biodegradable within the time period over whichrelease is desired or relatively soon thereafter, generally in the rangeof one year, more typically a few months, even more typically a few daysto a few weeks. Biodegradation can refer to either a breakup of themicroparticle, that is, dissociation of the polymers forming themicroparticles and/or of the polymers themselves. This can occur as aresult of change in pH from the carrier in which the particles areadministered to the pH at the site of release, as in the case of thediketopiperazines, hydrolysis, as in the case of poly(hydroxy acids), bydiffusion of an ion such as calcium out of the microparticle, as in thecase of microparticles formed by ionic bonding of a polymer such asalginate, and by enzymatic action, as in the case of many of thepolysaccharides and proteins. In some cases linear release may be mostuseful, although in others a pulse release or “bulk release” mayprovided more effective results.

Representative synthetic materials are: diketopiperazines, poly(hydroxyacids) such as poly(lactic acid), poly(glycolic acid) and copolymersthereof, polyanhydrides, polyesters such as polyorthoesters, polyamides,polycarbonates, polyalkylenes such as polyethylene, polypropylene,poly(ethylene glycol), poly(ethylene oxide), poly(ethyleneterephthalate), poly vinyl compounds such as polyvinyl alcohols,polyvinyl ethers, polyvinyl esters, polyvinyl halides,polyvinylpyrrolidone, polyvinylacetate, and poly vinyl chloride,polystyrene, polysiloxanes, polymers of acrylic and methacrylic acidsincluding poly(methyl methacrylate), poly(ethyl methacrylate),poly(butylmethacrylate), poly(isobutyl methacrylate),poly(hexylmethacrylate), poly(isodecyl methacrylate), poly(laurylmethacrylate), poly(phenyl methacrylate), poly(methyl acrylate),poly(isopropyl acrylate), poly(isobutyl acrylate), poly(octadecylacrylate), polyurethanes and co-polymers thereof, celluloses includingalkyl cellulose, hydroxyalkyl celluloses, cellulose ethers, celluloseesters, nitro celluloses, methyl cellulose, ethyl cellulose,hydroxypropyl cellulose, hydroxy-propyl methyl cellulose, hydroxybutylmethyl cellulose, cellulose acetate, cellulose propionate, celluloseacetate butyrate, cellulose acetate phthalate, carboxylethyl cellulose,cellulose triacetate, and cellulose sulphate sodium salt, poly(buticacid), poly(valeric acid), and poly(lactide-co-caprolactone).

Natural polymers include alginate and other polysaccharides includingdextran and cellulose, collagen, albumin and other hydrophilic proteins,zein and other prolamines and hydrophobic proteins, copolymers andmixtures thereof. As used herein, chemical derivatives thereof refer tosubstitutions, additions of chemical groups, for example, alkyl,alkylene, hydroxylations, oxidations, and other modifications routinelymade by those skilled in the art.

Bioadhesive polymers include bioerodible hydrogels described by H. S.Sawhney, C. P. Pathak and J. A. Hubell in Macromolecules, 1993, 26,581-587, polyhyaluronic acids, casein, gelatin, glutin, polyanhydrides,polyacrylic acid, alginate, chitosan, and polyacrylates.

A system based upon diketopiperazine structural elements or one of itssubstitution derivatives, including diketomorpholines, diketodioxanes orothers, forms microparticles with desirable size distributions and pHranges as well as good cargo tolerance. A wide range of stable,reproducible characteristics can be generated with appropriatemanipulations of the attachment sites, resulting in substantial yieldsand excellent reproducibility.

The diketopiperazines or their substitution analogs are rigid planarrings with at least six ring atoms containing heteroatoms and unbondedelectron pairs. One or both of the nitrogens can be replaced with oxygento create the substitution analogs diketomorpholine and diketodioxane,respectively. Although it is possible to replace a nitrogen with asulfur atom, this does not yield a stable structure. The general formulafor diketopiperazine and its analogs is shown below.

Wherein n is between 0 and 7, Q is, independently, a C₁₋₂₀ straight,branched or cyclic alkyl, aralkyl, alkaryl, alkenyl, alkynyl,heteroalkyl, heterocyclic, alkyl-heterocyclic, or heterocyclic-alkyl; Tis —C(O)O, —OC(O), —C(O)NH, —NH, —NQ, —OQO, —O, —NHC(O), —OP(O), —P(O)O,—OP(O)₂, —P(O)₂O, —OS(O)₂, or —S(O)₃; U is an acid group, such as acarboxylic acid, phosphoric acid, phosphonic acid and sulfonic acid, ora basic group, such as primary, secondary and tertiary amines,quaternary ammonium salts, guanidine, aniline, heterocyclic derivatives,such as pyridine and morpholine, or a zwitterionic C₁₋₂₀ chaincontaining at least one acidic group and at least one basic group, forexample, those described above, wherein the side chains can be furtherfunctionalized with an alkene or alkyne group at any position, one ormore of the carbons on the side chain can be replaced with an oxygen,for example, to provide short polyethylene glycol chains, one or more ofthe carbons can be functionalized with an acidic or basic group, asdescribed above, and wherein the ring atoms X at positions 1 and 4 areeither O or N.

As used herein, “side chains” are defined as Q-T-Q-U or Q-U, wherein Q,T, and U are defined above.

Examples of acidic side chains include, but are not limited, to cis andtrans —CH═CH—CO₂H, —CH(CH₃)═CH(CH₃)—CO₂H, —(CH₂)₃—CO₂H,—CH₂CH(CH₃)—CO₂H, —CH(CH₂CO₂H)═CH₂, -(tetrafluoro) benzoic acid,-benzoic acid and —CH(NHC(O)CF₃)—CH₂—CO₂H.

Examples of basic side chains include, but are not limited to, -aniline,-phenyl-C(NH)NH₂, -phenyl-C(NH)NH(alkyl), -phenyl-C(NH)N(alkyl)₂ and—(CH₂)₄NHC(O)CH(NH₂)CH(NH₂)CO₂H.

Examples of zwitterionic side chains include, but are not limited to,—CH(NH₂)—CH₂—CO₂H and —NH(CH₂)₁₋₂₀CO₂H.

The term aralkyl refers to an aryl group with an alkyl substituent.

The term heterocyclic-alkyl refers to a heterocyclic group with an alkylsubstituent.

The term alkaryl refers to an alkyl group that has an aryl substituent.

The term alkyl-heterocyclic refers to an alkyl group that has aheterocyclic substituent.

The term alkene, as referred to herein, and unless otherwise specified,refers to an alkene group of C₂ to C₁₀, and specifically includes vinyland allyl.

The term alkyne, as referred to herein, and unless otherwise specified,refers to an alkyne group of C₂ to C₁₀.

As used herein, “diketopiperazines” includes diketopiperazines andderivatives and modifications thereof falling within the scope of theabove-general formula.

The fumaryl diketopiperazine is most preferred for pulmonaryapplications.

B. Methods for Manufacture

The matrices can be formed of the polymers other than thediketopiperazines by solvent evaporation, spray drying, solventextraction and other methods known to those skilled in the art. Methodsdeveloped for making microspheres for drug delivery are described in theliterature, for example, as described by Mathiowitz and Langer, J.Controlled Release 5, 13-22 (1987); Mathiowitz, et al., ReactivePolymers 6, 275-283 (1987); and Mathiowitz, et al., J. Appl. PolymerSci. 35, 755-774 (1988), the teachings of which are incorporated herein.The selection of the method depends on the polymer selection, the size,external morphology, and crystallinity that is desired, as described,for example, by Mathiowitz, et al., Scanning Microscopy 4, 329-340(1990); Mathiowitz, et al., J. Appl. Polymer Sci. 45, 125-134 (1992);and Benita, et al., J. Pharm. Sci. 73, 1721-1724 (1984), the teachingsof which are incorporated herein.

In solvent evaporation, described for example, in Mathiowitz, et al.,(1990), Benita, and U.S. Pat. No. 4,272,398 to Jaffe, the polymer isdissolved in a volatile organic solvent. The drug, either in solubleform or dispersed as fine particles, is added to the polymer solution,and the mixture is suspended in an aqueous phase that contains a surfaceactive agent such as poly(vinyl alcohol). The resulting emulsion isstirred until most of the organic solvent evaporates, leaving solidmicrospheres.

In general, the polymer can be dissolved in methylene chloride. Severaldifferent polymer concentrations can be used, for example, between 0.05and 0.20 g/ml. After loading the solution with drug, the solution issuspended in 200 ml of vigorously stirring distilled water containing 1%(w/v) poly(vinyl alcohol) (Sigma Chemical Co., St. Louis, Mo.). Afterfour hours of stirring, the organic solvent will have evaporated fromthe polymer, and the resulting microspheres will be washed with waterand dried overnight in a lyophilizer.

Microspheres with different sizes (1-1000 microns) and morphologies canbe obtained by this method which is useful for relatively stablepolymers such as polyesters and polystyrene. However, labile polymerssuch as polyanhydrides may degrade due to exposure to water. For thesepolymers, hot melt encapsulation and solvent removal may be preferred.

In hot melt encapsulation, the polymer is first melted and then mixedwith the solid particles of DNA, preferably sieved to less than 50 μm.The mixture is suspended in a non-miscible solvent such as silicon oiland, with continuous stirring, heated to 5° C. above the melting pointof the polymer. Once the emulsion is stabilized, it is cooled until thepolymer particles solidify. The resulting microspheres are washed bydecantation with petroleum ether to give a free-flowing powder.Microspheres with diameters between one and 1000 microns can be obtainedwith this method. The external surface of spheres prepared with thistechnique are usually smooth and dense. This procedure is useful withwater labile polymers, but is limited to use with polymers withmolecular weights between 1000 and 50000.

Solvent removal was primarily designed for use with polyanhydrides. Inthis method, the drug is dispersed or dissolved in a solution of aselected polymer in a volatile organic solvent like methylene chloride.The mixture is then suspended in oil, such as silicon oil, by stirring,to form an emulsion. Within 24 hours, the solvent diffuses into the oilphase and the emulsion droplets harden into solid polymer microspheres.Unlike solvent evaporation, this method can be used to make microspheresfrom polymers with high melting points and a wide range of molecularweights. Microspheres having a diameter between one and 300 microns canbe obtained with this procedure. The external morphology of the spheresis highly dependent on the type of polymer used.

In spray drying, the polymer is dissolved in an organic solvent such asmethylene chloride (0.04 g/ml). A known amount of active drug issuspended (if insoluble) or co-dissolved (if soluble) in the polymersolution. The solution or the dispersion is then spray-dried. Typicalprocess parameters for a mini-spray drier are as follows: polymerconcentration=0.04 g/ml, inlet temperature=24° C., outlet temperature=13to 15° C., aspirator setting=15, pump setting=10 ml/min, spray flow=600NLh⁻¹, and nozzle diameter=0.5 mm. Microspheres ranging in diameterbetween one and ten microns can be obtained with a morphology whichdepends on the selection of polymer.

Double walled microspheres can be prepared according to U.S. Pat. No.4,861,627 to Mathiowitz.

Hydrogel microspheres made of gel-type polymers such as alginate orpolyphosphazines or other dicarboxylic polymers can be prepared bydissolving the polymer in an aqueous solution, suspending the materialto be incorporated into the mixture, and extruding the polymer mixturethrough a microdroplet forming device, equipped with a nitrogen gas jet.The resulting microspheres fall into a slowly stirring, ionic hardeningbath, as described, for example, by Salib, et al., PharmazeutischeIndustrie 40-11A, 1230 (1978), the teachings of which are incorporatedherein. The advantage of this system is the ability to further modifythe surface of the microspheres by coating them with polycationicpolymers such as polylysine, after fabrication, for example, asdescribed by Lim, et al., J. Pharm. Sci. 70, 351-354 (1981). Forexample, in the case of alginate, a hydrogel can be formed by ionicallycrosslinking the alginate with calcium ions, then crosslinking the outersurface of the microparticle with a polycation such as polylysine, afterfabrication. The microsphere particle size will be controlled usingvarious size extruders, polymer flow rates and gas flow rates.

Chitosan microspheres can be prepared by dissolving the polymer inacidic solution and crosslinking with tripolyphosphate. For example,carboxymethylcellulose (CMC) microsphere are prepared by dissolving thepolymer in an acid solution and precipitating the microspheres with leadions. Alginate/polyethylene imide (PEI) can be prepared to reduce theamount of carboxyl groups on the alginate microcapsules.

Methods for Synthesis of the Diketopiperazines

Diketopiperazines can be formed by cyclodimerization of amino acid esterderivatives, as described by Katchalski, et al., J. Amer. Chem. Soc. 68,879-880 (1946), by cyclization of dipeptide ester derivatives, or bythermal dehydration of amino acid derivatives in high-boiling solvents,as described by Kopple, et al., J. Org. Chem. 33(2), 862-864 (1968), theteachings of which are incorporated herein.2,5-diketo-3,6-di(aminobutyl)piperazine (Katchalski et al. refer to thisas lysine anhydride) was prepared via cyclodimerization ofN-epsilon-P-L-lysine in molten phenol, similar to the Kopple method inJ. Org. Chem., followed by removal of the blocking (P)-groups with 4.3 MHBr in acetic acid. This route is preferred because it uses acommercially available starting material, it involves reactionconditions that are reported to preserve stereochemistry of the startingmaterials in the product and all steps can be easily scaled up formanufacture.

Diketomorpholine and diketooxetane derivatives can be prepared bystepwise cyclization in a manner similar to that disclosed inKatchalski, et al., J. Amer. Chem. Soc. 68, 879-880 (1946).

Diketopiperazines can be radiolabelled. Means for attaching radiolabelsare known to those skilled in the art. Radiolabelled diketopiperazinescan be prepared, for example, by reacting tritium gas with thosecompounds listed above that contain a double or triple bond. A carbon-14radiolabelled carbon can be incorporated into the side chain by using¹⁴C labelled precursors which are readily available. These radiolabelleddiketopiperazines can be detected in vivo after the resultingmicroparticles are administered to a subject.

Synthesis of Symmetrical Diketopiperazine Derivatives

The diketopiperazine derivatives are symmetrical when both side chainsare identical. The side chains can contain acidic groups, basic groups,or combinations thereof.

One example of a symmetrical diketopiperazine derivative is2,5-diketo-3,6-di(4-succinylaminobutyl)piperazine.2,5-diketo-3,6-di(aminobutyl)piperazine is exhaustively succinylatedwith succinic anhydride in mildly alkaline aqueous solution to yield aproduct which is readily soluble in weakly alkaline aqueous solution,but which is quite insoluble in acidic aqueous solutions. Whenconcentrated solutions of the compound in weakly alkaline media arerapidly acidified under appropriate conditions, the material separatesfrom the solution as microparticles.

Other preferred compounds can be obtained by replacing the succinylgroup(s) in the above compound with glutaryl, maleyl or fumaryl groups.

Unsymmetrical Diketopiperazine Derivatives Unsymmetric Deprotection of aSymmetrical Diketopiperazine Intermediate.

One method for preparing unsymmetrical diketopiperazine derivatives isto protect functional groups on the side chain, selectively deprotectone of the side chains, react the deprotected functional group to form afirst side chain, deprotect the second functional group, and react thedeprotected functional group to form a second side chain.

Diketopiperazine derivatives with protected acidic side chains, such ascyclo-Lys(P)Lys(P), wherein P is a benzyloxycarbonyl group, or otherprotecting group known to those skilled in the art, can be selectivelydeprotected. The protecting groups can be selectively cleaved by usinglimiting reagents, such as HBr in the case of the benzyloxycarbonylgroup, or fluoride ion in the case of silicon protecting groups, and byusing controlled time intervals. In this manner, reaction mixtures whichcontain unprotected, monoprotected and di-protected diketopiperazinederivatives can be obtained. These compounds have different solubilitiesin various solvents and pH ranges, and can be separated by selectiveprecipitation and removal. An appropriate solvent, for example, ether,can then be added to such reaction mixtures to precipitate all of thesematerials together. This can stop the deprotection reaction beforecompletion by removing the diketopiperazines from the reactants used todeprotect the protecting groups. By stirring the mixed precipitate withwater, both the partially and completely reacted species can bedissolved as salts in the aqueous medium. The unreacted startingmaterial can be removed by centrifugation or filtration. By adjustingthe pH of the aqueous solution to a weakly alkaline condition, theasymmetric monoprotected product containing a single protecting groupprecipitates from the solution, leaving the completely deprotectedmaterial in solution.

In the case of diketopiperazine derivatives with basic side chains, thebasic groups can also be selectively deprotected. As described above,the deprotection step can be stopped before completion, for example, byadding a suitable solvent to the reaction. By carefully adjusting thesolution pH, the diprotected derivative can be removed by filtration,leaving the partially and totally deprotected derivatives in solution.By adjusting the pH of the solution to a slightly acidic condition, themonoprotected derivative precipitates out of solution and can beisolated.

Zwitterionic diketopiperazine derivatives can also be selectivelydeprotected, as described above. In the last step, adjusting the pH to aslightly acidic condition precipitates the monoprotected compound with afree acidic group. Adjusting the pH to a slightly basic conditionprecipitates the monoprotected compound with a free basic group.

Limited removal of protecting groups by other mechanisms, including butnot limited to cleaving protecting groups that are cleaved byhydrogenation by using a limited amount of hydrogen gas in the presenceof palladium catalysts. The resulting product is also an asymmetricpartially deprotected diketopiperazine derivative. These derivatives canbe isolated essentially as described above.

The monoprotected diketopiperazine is reacted to produce adiketopiperazine with one sidechain and protecting group. Removal ofprotecting groups and coupling with other side chains yieldsunsymmetrically substituted diketopiperazines with a mix of acidic,basic, and zwitterionic sidechains.

Other materials that exhibit this response to pH can be obtained byfunctionalizing the amide ring nitrogens of the diketopiperazine ring.

Methods for Forming Microparticles and Encapsulating Drug

In one embodiment, drug is encapsulated within microparticles bydissolving a diketopiperazine with acidic side chains in bicarbonate orother basic solution, adding the drug in solution or suspension to beencapsulated, then precipitating the microparticle by adding acid, suchas 1 M citric acid.

In a second embodiment, drug is encapsulated within microparticles bydissolving a diketopiperazine with basic side chains in an acidicsolution, such as 1 M citric acid, adding the drug in solution orsuspension to be encapsulated, then precipitating the microparticle byadding bicarbonate or other basic solution.

In a third embodiment, drug is encapsulated within microparticles bydissolving a diketopiperazine with both acidic and basic side chains inan acidic or basic solution, adding the drug in solution or suspensionto be encapsulated, then precipitating the microparticle by neutralizingthe solution.

The microparticles can be stored in the dried state and suspended foradministration to a patient. In the first embodiment, the reconstitutedmicroparticles maintain their stability in an acidic medium anddissociate as the medium approaches physiological pH in the range ofbetween 6 and 14. In the second embodiment, suspended microparticlesmaintain their stability in a basic medium and dissociate at a pH ofbetween 0 and 6. In the third embodiment, the reconstitutedmicroparticles maintain their stability in an acidic or basic medium anddissociate as the medium approaches physiological pH in the range of pHbetween 6 and 8.

II. Selection and Incorporation of Drugs.

A. Drugs to be Incorporated.

Generally speaking, any form of drug can be delivered. Examples includesynthetic inorganic and organic compounds, proteins and peptides,polysaccharides and other sugars, lipids, and nucleic acid sequenceshaving therapeutic, prophylactic or diagnostic activities. The agents tobe incorporated can have a variety of biological activities, such asvasoactive agents, neuroactive agents, hormones, anticoagulants,immunomodulating agents, cytotoxic agents, antibiotics, antivirals,antisense, antigens, and antibodies. In some instances, the proteins maybe antibodies or antigens which otherwise would have to be administeredby injection to elicit an appropriate response. More particularly,compounds that can be encapsulated include insulin, heparin, calcitonin,felbarnate, parathyroid hormone and fragments thereof, growth hormone,erythropoietin, zidovudine (AZT), didanosine (DDI), granulocyte colonystimulating factor (G-CSF), lamotrigine, chorionic gonadotropinreleasing factor, luteinizing releasing hormone, .beta.-galactosidaseand Argatroban. Compounds with a wide range of molecular weight can beencapsulated, for example, between 100 and 500,000 grams per mole.

Natural Biological Molecules

Proteins are defined as consisting of 100 amino acid residues or more;peptide are less than 100 amino acid residues. Unless otherwise stated,the term protein refers to both proteins and peptides. Examples includeinsulin and other hormones. Polysaccharides, such as heparin, can alsobe administered.

The drug can be administered as an antigen, where the molecule isintended to elicit a protective immune response, especially against anagent that preferentially infects the lungs, such as mycoplasma,bacteria causing pneumonia, and respiratory synticial virus. In thesecases, it may also be useful to administer the drug in combination withan adjuvant, to increase the immune response to the antigen.

Nucleic Acid Sequences

Any genes that would be useful in replacing or supplementing a desiredfunction, or achieving a desired effect such as the inhibition of tumorgrowth, could be introduced using the matrices described herein. As usedherein, a “gene” is an isolated nucleic acid molecule of greater thanthirty nucleotides, preferably one hundred nucleotides or more, inlength. Examples of genes which replace or supplement function includethe genes encoding missing enzymes such as adenosine deaminase (ADA)which has been used in clinical trials to treat ADA deficiency andcofactors such as insulin and coagulation factor VIII. Genes whicheffect regulation can also be administered, alone or in combination witha gene supplementing or replacing a specific function. For example, agene encoding a protein which suppresses expression of a particularprotein-encoding gene, or vice versa, which induces expresses of aprotein-encoding gene, can be administered in the matrix. Examples ofgenes which are useful in stimulation of the immune response includeviral antigens and tumor antigens, as well as cytokines (tumor necrosisfactor) and inducers of cytokines (endotoxin), and variouspharmacological agents.

Other nucleic acid sequences that can be utilized include antisensemolecules which bind to complementary DNA to inhibit transcription,ribozyme molecules, and external guide sequences used to target cleavageby RNAase P, as described by S. Altman, et al., of Yale University.

As used herein, vectors are agents that transport the gene into targetedcells and include a promoter yielding expression of the gene in thecells into which it is delivered. Promoters can be general promoters,yielding expression in a variety of mammalian cells, or cell specific,or even nuclear versus cytoplasmic specific. These are known to thoseskilled in the art and can be constructed using standard molecularbiology protocols. Vectors increasing penetration, such as lipids,liposomes, lipid conjugate forming molecules, surfactants, and othermembrane permeability enhancing agents are commercially available andcan be delivered with the nucleic acid.

Imaging Agents

Imaging agents including metals, radioactive isotopes, radioopaqueagents, fluorescent dyes, and radiolucent agents can also beincorporated. Radioisotopes and radioopaque agents include gallium,technetium, indium, strontium, iodine, barium, and phosphorus.

B. Loading of Drug.

The range of loading of the drug to be delivered is typically betweenabout 0.01% and 90%, depending on the form and size of the drug to bedelivered and the target tissue. In the preferred embodiment usingdiketopiperazines, the preferred range is from 0.1% to 50% loading byweight of drug.

C. Pharmaceutical Compositions.

The microparticles can be suspended in any appropriate pharmaceuticalcarrier, such as saline, for administration to a patient. In the mostpreferred embodiment, the microparticles will be stored in dry orlyophilized form until immediately before administration. They can thenbe suspended in sufficient solution, for example an aqueous solution foradministration as an aerosol, or administered as a dry powder.

D. Targeted Administration.

The microparticles can be delivered to specific cells, especiallyphagocytic cells and organs. Phagocytic cells within the Peyer's patchesappear to selectively take up microparticles administered orally.Phagocytic cells of the reticuloendothelial system also take upmicroparticles when administered intravenously. Endocytosis of themicroparticles by macrophages in the lungs can be used to target themicroparticles to the spleen, bone marrow, liver and lymph nodes.

The microparticles can also be targeted by attachment of ligands whichspecifically or non-specifically bind to particular targets. Examples ofsuch ligands include antibodies and fragments including the variableregions, lectins, and hormones or other organic molecules havingreceptors on the surfaces of the target cells.

The charge or lipophilicity of the microparticle is used to change theproperties of the protein carrier. For example, the lipophilicity can bemodified by linking lipophilic groups to increase affinity of some drugsfor the microencapsulation system, thereby increasing drug cargocapacity. Other modifications can be made before or after formation ofthe microparticle, as long as the modification after formation does nothave a detrimental effect on the incorporated compound.

E. Storage of the Microparticles.

In the preferred embodiment, the microparticles are stored lyophilized.The dosage is determined by the amount of encapsulated drug, the rate ofrelease within the pulmonary system, and the pharmacokinetics of thecompound.

III. Delivery of Microparticles.

The microparticles can be delivered using a variety of methods, rangingfrom administration directly into the nasal passages so that some of theparticles reach the pulmonary system, to the use of a powderinstillation device, to the use of a catheter or tube reaching into thepulmonary tract. Dry powder inhalers are commercially available,although those using hydrocarbon propellants are no longer used andthose relying on the intake of a breath by a patient can result in avariable dose.

FIG. 1 is a schematic of a preferred dry powder—breathactivated—compressed air, manually operated inhaler 10. A gel cap 12, orother container which is easily ruptured, filled with the proper doseand volume of microparticulates containing drug to be delivered, or themicroparticulates per se, 14 is inserted into the top 16 of the chamberto make an air seal. Manual activation of the plunger 18 by the patientruptures the gel cap 12, allowing its contents to fall into thereservoir 20. When the patient inhales, this causes the hinged, breathactivated valve 22 to open, releasing the pressurized air into thepowder reservoir 20, dispersing the powder 24 into the air stream at theoptimum moment in the inhalation cycle. The air pump 26 is manuallyoperated by the patient to fill the air reservoir 28 to a pre-determinedpressure. Further pumping by the patient produces an audible whistlethrough the relief valve 30, indicating that the inhaler 10 is ready foruse.

In a variation of this device, the gel cap 12 and plunger 18 is replacedwith a sealable opening which allows for insertion of a metered amountof powder or microparticulates into the reservoir.

The present invention will be further understood by reference to thefollowing non-limiting examples.

Example 1 Administration of Calcitonin-diketopiperazine Microparticlesto Sheep

Materials and Methods

Animals

Sheep (approximately 100 kg) were housed at SUNY Stony Brook HealthSciences Center. Animals were anesthetized with acepromazine/thiopentalsodium. A suspense of diketopiperazine microparticle/sCT (10.04 mg/mL in0.9% saline) was instilled in each lung of each sheep. A solution of sCT(10 mg/mL in 0.9% saline) was administered by subcutaneous injection tocontrol animals. An intravenous catheter was placed in the cephalic veinof each anesthetized animal for blood sampling.

Dosing Solutions and Calculations

The following dosing solutions were prepared on the day of theexperiment. The volume of the dose delivered was calculated based on theactual weight of the animal as described below.

Suspension: Microparticle/sCT [2.47% sCT by weight] 10.04 mg/mb in 0.9%saline (pH 5.5) Solution: 10 mg/mb sCT in 0.9% saline (pH 5.5)Calculation: Actual volume per sheep = (Wt subject/100 kg) × Vol/sheepBlood Sampling and Processing

Blood samples were drawn at the following time points: −5, 0 (predose),1, 3, 5, 10, 15, 30, 45, 60, 90 minutes, 2, 4, and 6 hours. Bloodsamples (3 mL) were collected by the intravenous line which was washedwith 0.9% saline containing heparin following each sample. Samples werecollected in heparinized vacutainer tubes (Becton Dickinson #6387Vacutainer Tubes (45 units USP sodium heparin), 3 mL draw, 64×10.25 mm]and placed on ice until processing. Tubes were centrifuged at 2000 rpmfor 15 minutes and plasma harvested immediately. Plasma was frozen ondry ice and stored at −40° C. until analysis.

Sample Analysis

Plasma sCT concentration was determined using radioimmunoassay (Researchand Diagnostic Antibodies, Berkeley, Calif., catalog #K-1235).

Study Design

The pharmacokinetics of sCT in sheep was determined after lunginstillation of a formulation of sCT in diketopiperazine microparticle(2.47% by weight sCT) at a sCT dose of 100 μg/kg. The results werecompared with 100 μg/kg sCT administered by subcutaneous injection as asolution in 0.9% saline. This study consisted of a crossover experimentin which each sheep was administered a suspension of diketopiperazinemicroparticles/sCT by instillation in the lung. One week later eachsheep received a subcutaneous injection of sCT in 0.9% saline at thesame dose.

Results

The mean pharmacokinetic profiles of plasma sCT delivered to the lungand subcutaneously were determined by radioimmunoassay. The absorptionin both the pulmonary and subcutaneous administrations was rapid withsignificant blood levels appearing within 30 minutes.

The truncated AUC (0 to 6 hours) was calculated for each animal usingthe trapezoidal rule. The bioavailability (F %) for each animal wascalculated from the ration of the AUCs for the pulmonary andsubcutaneous administrations. The mean (±standard error of the mean) AUCfor the pulmonary and subcutaneous administration was 6,505±1,443pg.hr/mL and 38,842±5,743 pg.hr/mL, respectively. The mean (±standarderror of the mean) of the individual animal bioavailabilities was 17±4%.

The mean (±standard error of the mean) peak plasma sCT level followingpulmonary administration was 2,864±856 pg/mL and occurred at 0.25 hours.The mean peak level following subcutaneous administration was9,407±1,792 pg/mL and occurred at 2 hours. The peak sCT level for thepulmonary administration was 32% of that for the subcutaneousadministration.

These results are shown in FIGS. 2 a and 2 b.

Discussion

These results show that sCT is absorbed into the blood stream whenadministered to the lung of sheep using the diketopiperazinemicroparticle formulation. The plot of the mean plasma sCT levels forthe pulmonary and subcutaneous administrations of sCT shows that thesubject to subject variability in the routes of delivery is comparable.The bioavailability of the pulmonary route in this preliminary analysiswas 17% versus the subcutaneous administration.

These data clearly show that sCT is absorbed when administered to thelung in a diketopiperazine microparticle formulation.

Modifications and variations of the compositions and methods of thepresent invention will be obvious to those skilled in the art from theforegoing detailed description. Such modifications and variations areintended to come within the scope of the appended claims.

1. A method for delivery of an active agent to the pulmonary systemcomprising: administering the active agent by inhalation of adiketopiperazine composition to a patient in need of treatment, whereinan effective amount of a dry powder comprising microparticles whichcomprise the diketopiperazine composition is administered to the patientusing air as a carrier, wherein the diketopiperazine compositioncomprises a diketopiperazine and the active agent, and wherein themicroparticles have a diameter between 0.5 microns and ten microns,wherein the microparticles are administered from a dry powder inhaler orfrom a container for a dry powder inhaler; and wherein the active agentis released from the microparticle at a pH of 6.0 or greater.
 2. Themethod of claim 1, wherein the diketopiperazine has the formula2,5-diketo-3,6-di(4-X-aminobutyl)piperazine, wherein X is selected fromthe group consisting of succinyl, glutaryl, maleyl, and fumaryl.
 3. Themethod of claim 2, wherein X is fumaryl.
 4. The method of claim 1,wherein the active agent is a therapeutic agent selected from the groupconsisting of insulin, calcitonin, felbamate, heparin, parathyroidhormone and fragments thereof, growth hormone, erythropoietin,zidovudine (AZT), didanosine (DDI), granulocyte colony stimulatingfactor (G-CSF), lamotrigine, chorionic gonadotropin releasing factor,luteinizing releasing hormone, β-galactosidase, and Argatrohan.
 5. Amicroparticulate system for drug delivery to the pulmonary systemcomprising: an inhaler component containing a diketopiperazinecomposition; wherein the inhaler component is a dry powder inhaler or acontainer for a dry powder inhaler; wherein the diketopiperazinecomposition is a dry powder comprising microparticles having a sizerange of between 0.5 and ten microns, wherein the microparticlescomprise an effective amount of a drug to be delivered and adiketopiperazine, and wherein the microparticles release the drug at apH of 6.0 or greater, in a pharmaceutically acceptable carrier foradministration to the lungs, wherein the carrier is air.
 6. The systemof claim 5, wherein the microparticles consist essentially of the drugand the diketopiperazine.
 7. The system of claim 5, wherein thediketopiperazine has the formula2,5-diketo-3,6-di(4-X-aminobutyl)piperazine wherein X is selected fromthe group consisting of succinyl, glutaryl, maleyl, and fumaryl.
 8. Thesystem of claim 7, wherein X is fumaryl.
 9. The system of claim 5,wherein the drug is selected from the group consisting of insulin,calcitonin, felbamate, heparin, parathyroid hormone and fragmentsthereof, growth hormone, erythropoietin, zidovudine (AZT), didanosine(DDI), granulocyte colony stimulating factor (C-CSF), lamotrigine,chorionic gonadotropin releasing factor, luteinizing releasing hormone,β-galactosidase, and Argatroban.
 10. The system of claim 9, wherein thedrug is insulin.
 11. The method of claim 4, wherein the active agent isinsulin.